In frog red blood cells, K-Cl cotransport (i.e., the difference between ouabain-resistant K fluxes in Cl and NO3) has been shown to mediate a large fraction of the total K+ transport. In the present study, Cl−-dependent and Cl−-independent K+ fluxes via frog erythrocyte membranes were investigated as a function of external and internal K+ ([K+] e and [K+] i ) concentration. The dependence of ouabain-resistant Cl−-dependent K+ (86Rb) influx on [K+] e over the range 0–20 mm fitted the Michaelis-Menten equation, with an apparent affinity (K m ) of 8.2 ± 1.3 mm and maximal velocity (V max ) of 10.4 ± 1.6 mmol/l cells/hr under isotonic conditions. Hypotonic stimulation of the Cl−-dependent K+ influx increased both K m (12.8 ± 1.7 mm, P < 0.05) and V max (20.2 ± 2.9 mmol/l/hr, P < 0.001). Raising [K+] e above 20 mm in isotonic media significantly reduced the Cl−-dependent K+ influx due to a reciprocal decrease of the external Na+ ([Na+] e ) concentration below 50 mm. Replacing [Na+] e by NMDG+ markedly decreased V max (3.2 ± 0.7 mmol/l/hr, P < 0.001) and increased K m (15.7 ± 2.1 mm, P < 0.03) of Cl−-dependent K+ influx. Moreover, NMDG+ Cl substitution for NaCl in isotonic and hypotonic media containing 10 mm RbCl significantly reduced both Rb+ uptake and K+ loss from red cells. Cell swelling did not affect the Na+-dependent changes in Rb+ uptake and K+ loss. In a nominally K+(Rb+)-free medium, net K+ loss was reduced after lowering [Na+] e below 50 mm. These results indicate that over 50 mm [Na+] e is required for complete activation of the K-Cl cotransporter. In nystatin-pretreated cells with various intracellular K+, Cl−-dependent K+ loss in K+-free media was a linear function of [K+] i , with a rate constant of 0.11 ± 0.01 and 0.18 ± 0.008 hr−1 (P < 0.001) in isotonic and hypotonic media, respectively. Thus K-Cl cotransport in frog erythrocytes exhibits a strong asymmetry with respect to transported K+ ions. The residual, ouabain-resistant K+ fluxes in NO3 were only 5–10% of the total and were well fitted to linear regressions. The rate constants for the residual influxes were not different from those for K+ effluxes in isotonic (∼0.014 hr−1) and hypotonic (∼0.022 hr−1) media, but cell swelling resulted in a significant increase in the rate constants.
The Journal of Membrane Biology – Springer Journals
Published: Dec 1, 1999
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